Effects Of Amino Acid Nutrient Deprivation On The Regulation Of Cellular LDL Uptake

BackgroundCardiovascular and cerebrovascular diseases are one of the major diseases that currently affect human health and consume a large amount of social medical resources worldwide.According to statistics,about 3 million people die of cardiocerebral and vascular diseases every year in China.Atherosclerosis(AS)is one of the main causes of cardiovascular and cerebrovascular diseases.Plasma Low Density Lipoprotein Chesterol(LDL-C)levels are positively correlated with AS risk.LDL receptor(LDLR)is closely related to LDL metabolism in the body.The classical endocytosis pathway mediated by LDLR is the main way to clear LDL in vivo.At present,statins that are widely used in clinical practice can effectively prevent the occurrence of cardiovascular and cerebrovascular diseases by regulating the expression of LDLR and reducing the level of LDL-C in the blood.However,in FH patients,these drugs cannot achieve the reduction of LDL-C.And cardiovascular risk,about 30%of patients are intolerant to statins.Therefore,it is still of great practical significance to explore and discover new therapeutic targets and drugs.Amino acid homeostasis plays an important role in regulating the biological function of cells.Studies have shown that amino acid starvation(AAS)can induce autophagy by acting on mTORCl signaling pathway,but whether AAS can regulate LDL uptake has not been reported.In this project,we will study the effect of AAS on the endocytosis of LDL and its possible mechanism.Objective1.To study the effect of AAS on LDL uptake2.Study the effect of AAS on the expression and spatial distribution of LDLR3.Study the possible mechanism of AAS regulating LDL uptakeMethods1.In mouse embryonic fibroblasts(NIH-3T3)and human hepatocarcinoma cells(HepG2),the effect of AAS stimulation on LDL uptake was studied,and then the results of the experiment were reverse verified by complementing the missing amino acid components in the amino acid deficient medium.The cells were treated with clathrin inhibitors(pitstop2 and dynasore).The changes of Dil-LDL uptake were detected by confocal laser microscopy.To observe the effect of low glucose and serum on LDL uptake.WT mice and LDLR-/-mice of 8 weeks old were selected to extract liver primary cells,cultured in vitro and detected the effect of AAS on Dil-LDL uptake by fluorescence microscope.2.Actinomycin D and Cycloheximide treatment group had no significant effect on the uptake of LDL by cells.Western Blot and PCR results showed that AAS stimulation had no significant effect on LDLR expression.Flow cytometry and immunofluorescence showed that LDLR expression on the membrane surface increased after AAS.Real-time quantitative PCR and Western Blot results showed that transient AAS did not affect the expression of p62 and LC3B,there was no strict time correlation between AAS-induced LDL uptake and autophagy.We treated the cells with Rapamycin,an inducer of autophagy,and the results showed that Rapamycin had no significant effect on the uptake of LDL,that is,the activation of autophagy did not mimic the effect of AAS.At the same time,we transfected ATG5 siRNA to inhibit autophagy,confocal microscopy results showed that ATG5 siRNA had no significant effect on the uptake of Dil-LDL.3.Cells were transfected with the plasmid pEGFP-LDLR,and fluorescence reeovery after photobleaching(FRAP)was used to observe the effect of AAS on the movement rate of LDLR molecules.4.The cells were treated with PI3K inhibitor wortmannin,AKT kinase inhibitor AKT1/2,VPS34 inhibitor VPS34-IN1,SAR405,and laser confocal microscopy was used to detect changes in Dil-LDL uptake by cells.Compare the effect of AAS on cellular uptake of Dil-LDL with the effect of pravastatin.Results1.In NIH-3T3 and HepG2 cells,amino acid starvation was administered at different times.The results of laser confocal microscopy showed that the cells increased the uptake of Dil-LDL in a time-dependent manner and the phenomenon was reversed after supplementation of amino acids.Low glucose and low serum had no effect on LDL uptake.Clathrin inhibitors inhibit the effect of AAS on increasing LDL uptake,and in LDLR-/-mice,AAS has no significant effect on LDL uptake2.Actinomycin D and Cycloheximide treatment had no significant effect on LDL uptake by cells.Western Blot and PCR results showed that AAS stimulation had no significant effect on LDLR expression.Flow cytometry and immunofluorescence showed that LDLR expression on the membrane surface increased after AAS.Real time quantitative PCR and Western Blot results showed that transient AAS did not affect the expression of p62 and LC3B.Autophagy inducer Rapamycin treatment had no significant effect on LDL uptake.At the same time,ATG5 siRNA-transfected cells inhibited autophagy.Confocal microscopy results showed that ATG5 siRNA had no significant effect on the uptake of Dil-LDL.3.FRAP experiment results show that AAS shortens the time required for fluorescence recovery in the bleached area,suggesting that AAS increases the speed of LDLR movement,which in turn increases LDL transport.4.The results of laser confocal microscopy showed that AKT1/2 inhibitor had no effect on AAS.SAR405,VPS34-IN1 and wortmannin significantly reduced the effect of AAS on the uptake of Dil-LDL by cells.Within a short time(4h),both AAS and Pravastatin increased Dil-LDL uptake.AAS promoted Dil-LDL uptake similar to statins.Conclusions1.AAS induced cells increase LDL uptake2.The role of AAS in increasing LDL uptake by cells is dependent on clathrin pathway and LDLR3.AAS increases the distribution of LDLR on the surface of cell membranes instead of affecting its expression4.The role of AAS in increasing LDL uptake may be partially dependent on VPS34OutlookFurther studies on the signal transduction pathways that AAS affects the spatial distribution of LDLR may provide new ideas for developing lipid-lowering methods that do not depend on regulating LDLR gene expression.